Abstract: Problems in protein folding and aggregation are the root cause of many of the most devastating diseases, challenging public health worldwide. Therefore, it is even more devastating to understand that there is currently no treatment for these neurodegenerative diseases-in many cases not even for reliably delaying symptoms. It is safe to say that much of the molecular mechanisms by which these proteins begin to aggregate, form soluble oligomers and elongate to fiber are not understood, nor is the debate settled whether the cause of neurotoxicity is the loss of the original function of the protein that is being recruited for aggregation, the soluble oligomer that may permeate through and destroy cell membranes, the suffocation of important biological space by fibrous aggregates, or the combination of these or other factors. A key challenge for tackling these questions is the lack of physical tools that can transiently characterize early protein aggregation events, including protein conformational changes and the formation of soluble protein oligomers, and can do so with site-specificity, transiently, in the presence of key biological constituents, and ultimately in vivo. I propose to develop a set of innovative instrumentations and novel approaches for enhancing the sensitivity and selectivity of magnetic resonance detection by more than two orders of magnitude compared to existing nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) experiments. Our target is the time-resolved visualization of molecular interfaces during protein aggregation, in the presence of lipid membranes and as a function of mutation and chemical signals, e.g. of molecular chaperones and osmolytes. We will accompany our in vitro studies with probing protein aggregation events in live cells and tracking live cell survivability. These studies are made possible with selective spin labeling of interfacial protein sites and the sensitive detection of transient molecular interactions, through the measurements of interfacial hydration water dynamics that is sensitively modulated upon molecular approach within distant 10 [unreadable] of spin labeled sites, as well as through the enhanced measurement of electron spin-spin distances and dynamics. I will achieve the former innovation with the novel Overhauser dynamic nuclear polarization technique, and the latter with unprecedented pulse shaping capability to significantly enhance pulsed EPR performance. I will focus in the first years on the study of tau whose fibrous tangles are found in Alzheimer's and other neurodegenerative diseases. We will work with the two full length human isoforms, tau-3R and tau-4R, as well as the caspase-cleaved tau proteins, as has been recently found to precede tangle formation in vivo. The strength of our tools and strategies is that they are generally applicable to all aggregating proteins implicated in neurodegenerative diseases. Our proposed studies focus on unraveling the roles of protein oligomers and aggregates in disease-related effects, and pursue to address a significant biomedical problem: the development of rational treatments and earlier diagnosis for neurodegenerative diseases. Public Health Relevance: I propose to develop a set of innovative instrumentations and methods for enhancing the sensitivity of conventional magnetic resonance spectroscopic tools by more than two orders of magnitude that will allow for unprecedented studies of early protein aggregation mechanisms that are largely invisible to existing techniques. This will lead to unraveling the relationships of misfolded proteins, oligomers or fibrous deposits to disease-related effects, and addresses a significant biomedical problem of developing rational treatments and earlier diagnosis strategies for neurodegenerative diseases such as Alzheimer's disease.